Template:Kyoto/Notebook/Sep 22

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Sep 22

Colony PCR

Tatsui

Sample	base pair
9/20 Plac-PT181 attenuator(1~4)	664
9/20 Plac-PT181 antisense(1~6)	369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)	859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)	723

Miniprep

Honda

DNA	concentration[µg/mL]	260/280	260/230
9/21 spinach-DT	197.9	1.67	1.54
9/21 Plux	125.9	1.71	1.27
9/21 Plux-RBS-GFP-DT-(1)	168.0	1.62	1.73
9/21 Pcon-PT181 attenuator-(1)	368.3	1.57	1.62
9/21 RBS-GFP-DT	532.8	1.75	1.78
9/21 Pcon-RBS-GFP-DT	845.2	2.02	1.70

Colony PCR

Honda

Sample	base pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min10s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-aptamer12-1R-DT(E-1A)	--
6~8	Pcon-spinach-DT(E-1A)	--
10~12	Pcon-attenuator PT181(E-1A)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	DT(1-3)	--
6~8	aptamer12-1RDT(1-3)	--
10~12	Pcon-PT181 attenuator(1-5A)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-aptamer12-1R-DT(E-1A)	31.2	1.87	0.30
Pcon-spinach-DT(E-1A)	25.6	1.88	0.99
Pcon-attenuator PT181(E-1A)	25.3	1.99	0.86
DT(1-3)	22.1	1.82	0.20
aptamer12-1RDT(1-3)	33.5	1.99	0.05
Pcon-PT181 attenuator(1-5A)	36.9	1.12	0.96

digestion

No name

	9/16 Plac	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	12.5µL	0µL	1µL	1µL	3µL	3µL	9.5µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/22 Plux	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	16µL	0µL	1µL	1µL	3µL	3µL	14µL	30µL
NC	0.8µL	0µL		0µL	0µL	1µL	1µL	7.2µL	10µL

	9/20 RBS-tetR-2R-DT	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	8.5µL	1µL	0µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
2	100bp ladder	--
4~6	Pcon-attenuator PT181(2-SA)	--

Lane	DNA	Enzyme
1	100bp ladder	--
3~5	spinach-DT(3-2)	--
8~10	Ptet-antisense PT181(1-3)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator PT181(E-0)	2.4	1.80	0.17
Pcon-attenuator PT181(2-SA)	10.8	1.85	0.54
spinach-DT(3-2)	36.7	1.80	0.75
Ptet-antisense PT181(1-3)	16.8	1.74	0.78

Colony PCR

No name

Sample	base pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)	859

Colony PCR

Tatsui

Sample	base pair
PconPT181antisense-spinach-DT(9~15)	723

Colony PCR

No name

Sample	base pair
pSB1C3+P-KaiBC①	283
pSB1C3+P-KaiBC②	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
2min	30s	30s	18s	30cycles

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/18 Plac (SpeI & PstI)	3.0 µL	9/18 RBS-lysis3-DT (XbaI & PstI)	12.4 µL	3.5 µL
experiment	9/12 Ptet-pT181 antisense	1.0 µL	9/18 spinach-DT	11.9 µL	3.5 µL
experiment	9/13 Pcon-RBS-luxR-DT	2.1 µL	9/10 Plux-RBS-GFP-DT	5.6 µL	3.5 µL
experiment	9/13 Pcon-pT181 attenuator (SpeI & PstI)	2.0 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	6.3 µL	3.5 µL
experiment	9/18 Plac ②(SpeI & PstI)	3.0 µL	9/18 aptamer 12-1R-DT(XbaI & PstI)	4.5µL	x3.5 µL

Liquid Culture

No name

Sample	medium
9/2 spinach-DT-2	--
8/16 Plux-4	--
8/27 Plux-RBS-GFP-DT-1	--
9/5 Pcon-pT181 attenuator-1	--

digestion

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	100bp ladder	--
3	RBS-lacZα-DT	XbaI&PstI
4	RBS-lacZα-DT	XbaI&PstI
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(XbaI&PstI)	9.8	1.50	0.39

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

PCR

Tatsui

Sample	base pair
pT181 attenuator(pSB1C3)	--
Pcon-RBS-lacZα-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	3 min	30 cycles

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

Contents

Sep 22

Colony PCR

Miniprep

Colony PCR

Gel Extraction

digestion

Gel Extraction

Colony PCR

Colony PCR

Colony PCR

Ligation

Liquid Culture

digestion

Gel Extraction

Transformation

PCR

Transformation